CN102242073A - Method for preparing daptomycin by fermentation - Google Patents
Method for preparing daptomycin by fermentation Download PDFInfo
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- CN102242073A CN102242073A CN 201010168925 CN201010168925A CN102242073A CN 102242073 A CN102242073 A CN 102242073A CN 201010168925 CN201010168925 CN 201010168925 CN 201010168925 A CN201010168925 A CN 201010168925A CN 102242073 A CN102242073 A CN 102242073A
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- Prior art keywords
- daptomycin
- fermentation
- value
- deionized water
- capric acid
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Links
- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 69
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 title claims abstract description 69
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 67
- 238000000855 fermentation Methods 0.000 title claims abstract description 55
- 230000004151 fermentation Effects 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 29
- 241000958215 Streptomyces filamentosus Species 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 6
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 claims description 53
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000008367 deionised water Substances 0.000 claims description 22
- 229910021641 deionized water Inorganic materials 0.000 claims description 22
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 239000002518 antifoaming agent Substances 0.000 claims description 14
- 229940041514 candida albicans extract Drugs 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 14
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 14
- 238000011081 inoculation Methods 0.000 claims description 14
- 229920001296 polysiloxane Polymers 0.000 claims description 14
- 239000012138 yeast extract Substances 0.000 claims description 14
- 239000004375 Dextrin Substances 0.000 claims description 7
- 229920001353 Dextrin Polymers 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 7
- 235000010469 Glycine max Nutrition 0.000 claims description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 7
- NEEHYRZPVYRGPP-IYEMJOQQSA-L calcium gluconate Chemical compound [Ca+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O NEEHYRZPVYRGPP-IYEMJOQQSA-L 0.000 claims description 7
- 235000019425 dextrin Nutrition 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 108010050327 trypticase-soy broth Proteins 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 5
- 230000033228 biological regulation Effects 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 2
- 230000004927 fusion Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 238000005215 recombination Methods 0.000 abstract description 2
- 238000010364 biochemical engineering Methods 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 230000006798 recombination Effects 0.000 abstract 1
- 239000002243 precursor Substances 0.000 description 24
- 230000012010 growth Effects 0.000 description 19
- 239000002253 acid Substances 0.000 description 17
- 241000196324 Embryophyta Species 0.000 description 16
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 12
- 241001052560 Thallis Species 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 241001655322 Streptomycetales Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
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- 239000000203 mixture Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 229940021392 cubicin Drugs 0.000 description 2
- -1 cyclic ester Chemical class 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- DOAKLVKFURWEDJ-OFNKPWESSA-N daptomycin Chemical compound C([C@H]1C(=O)OC(C)C(C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)C(C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-OFNKPWESSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000009123 feedback regulation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
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- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a daptomycin high-yield strain and a method for preparing daptomycin by fermentation, and belongs to the technical field of biochemical engineering. In the invention, streptomyces roseosporus is utilized as starting bacterium and is treated by protolast fusion and gene recombination processes to form a high-yield mutagenized strain WKL-126. The high-yield mutagenized strain WKL-126 is preserved in the China general microbiological culture collection center (CGMCC) at present and an accession number is 3731. The invention also discloses a method for preparing daptomycin through adopting the strain WKL-126. Compared with the prior art, the method can realize that a fermentation quantity per unit is over 2000 mg/L.
Description
Technical field
The present invention relates to a kind of daptomycin induced mutations superior strain, and the method that adopts this strain fermentation legal system to get daptomycin, technical field of biochemical industry belonged to.
Background technology
Daptomycin (Daptomycin) (trade(brand)name cubicin) is to extract a cyclic ester peptide matters that obtains from rose-colored spore streptomycete (Streptomycesroseosporus) fermented liquid.The daptomycin molecular formula is C
72H
101N
17O
26, molecular weight 1619.71, wherein 4 Threonines and 13 kynuric acids in 13 amino acid moleculars are closed into ring-type with peptide bond, and the side chain terminal amino acid tryptophan links to each other with amido linkage with capric acid, constitutes the acyclic acidic lipopeptide antibiotic.Its structural formula is as shown below:
Generally, daptomycin is yellow or light yellow crystal material, can be water-soluble, methyl alcohol, ethanol, propyl alcohol, propyl carbinol, dimethyl formamide, dichloro heterocycle ethane, tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO), slightly soluble or be insoluble to acetone, chloroform, ether, benzene and ethyl acetate.
In September, 2003, because patient is to the antibiotic active demand of novel resistance, FDA (FDA) is through quick trial program approval injection daptomycin (trade(brand)name Cubicin
) be used for the treatment of the concurrency skin and the skin texture infection that cause by some Gram-positive sensitive strains, infect and skin ulcer as abscess, operative incision.According to statistics, this medicine goes on the market and promptly obtained about 6,000 ten thousand dollars income in back 1 year.Although daptomycin is faced with inevitable competition situation, the whole market share is also relatively limited, because the resistance gram-positive microorganism has the trend of increased popularity, it still has deep potentiality in the several years in the future.Though daptomycin is also limited the use of work two, three-way medication as linwzolid and common bacteriocidin, but because it is effective to vancomycin resistance and the bacterial strain of anti-the linwzolid, so in fact it can also infect the roentgenism x medication from the institute district and tie up many shares, these additional purpose of daptomycin also can improve its sales volume in the future.The sales volume of daptomycin on institute district treatment of infection pharmaceutical market in 2007 reaches 200,000,000 dollars, also has rising potential afterwards in the several years.
At present, the external daptomycin of producing mainly contains two kinds of operational paths, the one, be precursor with A21978C, under the effect of soda acid or deacylase, slough the side chain terminal acyl group, make side chain terminal connect the capric acid acyl group again through multistep chemical reactions such as radical protection, acylations then, thereby the formation daptomycin, this processing step is many, environmental pollution is serious, product yield extremely low (about 10%), purity are not high; The 2nd, directly form daptomycin by rose-colored spore streptomycete (Streptomyces roseosporus) fermentation, but owing to multiple factors such as fermentation unit are lower, cause that daptomycin suitability for industrialized production difficulty is big, cost is high, thereby causing the high and output of price can not satisfy the demand of world market far away, is one of world market medical material in short supply.
Because commercial benefits, gordian techniquies such as some external bacterial classifications and technology are externally maintained secrecy, seldom open report.Lilly Co., Eli. carry out for many years optimization of fermentation condition and product extraction work, but product production is never than quantum jump since the separation eighties in last century obtains daptomycin production bacterium Streptomyces roseosporus.At home, although the existing bacterial classification of producing daptomycin, the ability that this bacterial strain is produced daptomycin a little less than, usefulness is low.Therefore, selecting bacterial strain that a plant height produces daptomycin produces and has bigger society and economic benefit realizing the daptomycin industrialization.
Summary of the invention
The technical issues that need to address of the present invention are the superior strains that disclose a kind of daptomycin, and the method that adopts this strain fermentation legal system to get daptomycin, to overcome the defective that fermentation period is long, unit output is low, transformation efficiency is not high in the existing daptomycin fermentation technique.
For addressing the above problem, the invention provides a kind of daptomycin superior strain WKL-126.This bacterial strain is to be the bacterium that sets out with rose-colored spore streptomycete, obtains by protoplastis fusion and gene recombination technology, and in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, preserving number is: CGMCCNo.3731 at present.Preservation is centered close to No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date was preserved 30 years from 04 14th, 2010.This strain classification name: Streptomyces roseosporus (Streptomyces roseosporus), WKL-126.
But the production level of microbial fermentation not only depends on the performance of bacterial classification itself, and will have suitable envrionment conditions that its throughput is fully showed.Therefore must understand the requirement of bacterial classification by the whole bag of tricks, and understand the metabolic regulation mechanism and the possible pathways metabolism of bacterial classification in the synthetic product process, for technology reasonable in design is provided fundamental basis to envrionment conditions.Fermention medium can be for growth, breeding and synthetic product.Fermention medium can ramp arrive certain mycelial concentration after should making the seed inoculation, make long good mycelia can synthesize required product rapidly again, therefore, the composition of fermention medium also will have required element-specific of product and promotor etc. except that necessary element of mycelial growth and compound are arranged.Because the difference of fermentation purposes, the composition difference of its substratum is also very big.Fermenting process is subjected to the influence of many factors and the restriction of processing condition, and influence factor mainly contains temperature, pH value, carbon source, nitrogenous source, dissolved oxygen, inorganic salt of fermentation etc.Our company carried out abundant research to the above-mentioned influence factor that influences the superior strain fermentation level, optimized the processing condition that the superior strain fermentation method is produced daptomycin.
In addition, in the fermentation method of daptomycin was produced, the interpolation concentration of precursor capric acid and interpolation time were key factors.Excessive precursor tends to the thalline toxigenicity or has the feedback regulation effect, and precursor adds not enough a large amount of purpose product that can't guarantee to synthesize.Daptomycin is a kind of typical microbial secondary meta-bolites, has 2 physiological stages, i.e. thalli growth stage and meta-bolites synthesis phase in secondary metabolism.Add precursor too early and be unfavorable for that thalli growth and the final product that influences generate, cross to add precursor late, one side is unfavorable for the generation of end product daptomycin because a large amount of parent nucleus that form can not the feedback inhibition effect can be occurred by acidylate.On the other hand, cross the slow precursor that adds and cause it under-supply, influence the generation of end product.When just beginning to form, parent nucleus adds the formation that precursor helps end product most; acylation reaction can take place with parent nucleus in the capric acid that adds this moment fast; simultaneously since this moment thalline be in the logarithmic phase middle and later periods, to having toxic precursor capric acid stronger tolerance is arranged.Therefore the present invention has optimized capric acid joining day, fermentation time etc. to be fit to needs of scale production.
The present invention is on above-mentioned research basis, provides to adopt the superior strain fermentation method to produce the method for daptomycin, has provided the composition of fermentation culture carbon source, nitrogenous source and other substratum optimized, optimizes dissolved oxygen.The technical scheme of this method is as follows:
Concrete fermentation condition is as follows:
The first order seed substratum: trypticase soya broth 2.0～4.0%, dextrin 2.0～3.0%, deionized water is supplied, and the pH value is 6.0～8.0.
Secondary seed medium: soyflour 0.4～0.5%, yeast extract 0.4～0.6%, calglucon 0.8～1.2%, KCl 0.01～0.03%, MgSO
47H
2O 0.01～0.03%, FeSO
47H
2O 0.0003～0.0005%, Sag 471 (silicone antifoam agent) 0.02～0.04%, and deionized water is supplied, and the pH value is 6.0～8.0.
Fermention medium is: glucose 0.80～0.85%, CaCO
30.1～0.3%, soyflour 1.0～3.0%, yeast extract 0.05～0.15%, Fe (NH
4)
2SO
46H
2O 0.065～0.068%, and KCl 0.015～0.025%, MgSO
47H
2O 0.015～0.025%, FeSO
47H
2O 0.0003～0.0005%, Sag 471 (silicone antifoam agent) 0.01～0.03%, and deionized water is supplied, and the pH value is 6.0～8.0.
The seed culture condition is: bacterial classification recovery back direct inoculation is in the 250mL Erlenmeyer flask that 50mL first order seed substratum is housed, and 30 ℃ of shaking table 250rpm cultivate 48h.Get 10mL first order seed substratum, be inoculated into the 500mL Erlenmeyer flask of 250mL secondary seed medium, 30 ℃ of shaking table 250rpm cultivate 24h.
Fermentation culture conditions is: liquid amount is 20～50%, and the inoculation volume is 2～5%, air air flow 1～5L/min, preceding 15h stirring intensity 220～280rpm, 350～450rpm then, 25～35 ℃ of culture temperature, fermentation back 10h by add ammoniacal liquor keep pH value 6.5 or more than.Since the 28th hour, with every liter of nutrient solution per hour the amount of 0.25～0.28mL add aseptic caproic acid (50%v/v), until results.
Capric acid stream adds CONTROL PROCESS: the interpolation time of capric acid and interpolation concentration (the capric acid concentration of measuring in the fermented liquid) are about 10～12h, 0.1～0.8mmol/L and 35～40h, 0.8～1.2mmol/L, adopt the method for fed-batch to carry out the regulation and control of tunning, cell concentration reaches and stops fermentation about 2000mg/L.
The concentration of daptomycin adopts high performance liquid chromatography (HPLC) method to determine in the fermented liquid:
(1) selection of chromatographic condition
According to the character and the chromatogram requirement of daptomycin, determine the operational condition of high performance liquid chromatography:
Stationary phase: Kromasil C18 chromatographic column (4.6mm * 250mm); Moving phase: the acetonitrile that contains 0.1% trifluoroacetic acid of 45: 54 (volume ratio): contain the water of 0.1% trifluoroacetic acid, flow velocity 1.0mL/min; 30 ℃ of column temperatures, the UV device detects wavelength 220nm.
(2) foundation of typical curve
The standard substance of daptomycin are mixed with the standardized solution of different concentration known with deionized water.Sample introduction 10 μ L under (1) described chromatographic condition measure with HPLC, carry out stratographic analysis.Peak area to analytical results utilizes chromatographic working station to carry out integration, and is the longitudinal axis with the integral result, is transverse axis with the concentration of sample, draws the typical curve of daptomycin.The linear regression process is finished automatically by chromatographic working station and the result is stored in the analytical procedure.
(3) foundation of typical curve
Get the 10ml fermentation broth sample, centrifugal 10min gets supernatant liquor under the 8000rpm, and centrifugal 5min under 8000rpm gets supernatant liquor again, 0.2 μ m micro-pore-film filtration.Get filtrate 10 μ L respectively, measure with HPLC.
Embodiment
Selected bacterial classification is artificial mutagenicity high-yield bacterial strain among the present invention, and in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, preserving number is: 3731 at present.Used peptone, the yeast powder of project is OXOID company product, and other are homemade analytical reagent.Understand content of the present invention in order better to help, the spy enumerates following example, but is not limited to the present invention.
Embodiment 1
(1) seed culture
The first order seed substratum: trypticase soya broth 2.0%, dextrin 2.0%, deionized water is supplied, and the pH value is 6.0.
Secondary seed medium: soyflour 0.4%, yeast extract 0.4%, calglucon 0.8%, KCl0.01%, MgSO
47H
2O 0.01%, FeSO
47H
2O 0.0003%, Sag 471 (silicone antifoam agent) 0.02%, and deionized water is supplied, and the pH value is 6.0.
Culture condition: bacterial classification recovery back direct inoculation is in the 250mL Erlenmeyer flask that 50mL first order seed substratum is housed, and 30 ℃ of shaking table 250rpm cultivate 48h.Get 10mL first order seed substratum, be inoculated into the 500mL Erlenmeyer flask of 250mL secondary seed medium, 30 ℃ of shaking table 250rpm cultivate 24h.
(2) fermentation culture
Fermention medium: glucose 0.85%, CaCO
30.3%, soyflour 3.0%, yeast extract 0.15%, Fe (NH
4)
2SO
46H
2O 0.068%, and KCl 0.025%, MgSO
47H
2O 0.025%, FeSO
47H
2O0.0005%, Sag 471 (silicone antifoam agent) 0.03%, deionized water is supplied, and the pH value is 6.0.
Fermentation culture conditions: liquid amount is 20%, and the inoculation volume is 2%, air air flow 1L/min, and preceding 15h stirring intensity 220rpm, 350rpm then, 25 ℃ of culture temperature, fermentation back 10h keeps pH value 6.5 by adding ammoniacal liquor.Since the 28th hour, with every liter of nutrient solution per hour the amount of 0.25mL add aseptic caproic acid (50%v/v), until results.
(3) capric acid stream adds
The thalli growth initial stage, medicine irritation was relatively more responsive to external world, add capric acid this moment and be unfavorable for that thalli growth and daptomycin synthesize, monitor by high performance liquid chromatography, add capric acid in the logarithmic growth early stage (10h) that the daptomycin parent nucleus has just produced, certain herbaceous plants with big flowers acid final concentration is 0.2mmol/L, allow thalline adapt to growing environment, relatively help the synthetic of later stage daptomycin.Yet, once add precursor and easily cause the precursor deficiency, can not continue a large amount of synthetic daptomycins, therefore (35h) adds certain herbaceous plants with big flowers acid when thalline enters logarithmic phase, and certain herbaceous plants with big flowers acid final concentration is 0.8mmol, guarantees the precursor consumption of mass production daptomycin.
The fermentation unit that records daptomycin in the fermented liquid after fermentation reaction finishes is 2001mg/L.
Embodiment 2
(1) seed culture
The first order seed substratum: trypticase soya broth 3.0%, dextrin 2.5%, deionized water is supplied, and the pH value is 7.0.
Secondary seed medium: soyflour 0.45%, yeast extract 0.5%, calglucon 0.9%, KCl0.02%, MgSO
47H
2O 0.02%, FeSO
47H
2O 0.0004%, Sag 471 (silicone antifoam agent) 0.03%, and deionized water is supplied, and the pH value is 7.0.
Culture condition: bacterial classification recovery back direct inoculation is in the 250mL Erlenmeyer flask that 50mL first order seed substratum is housed, and 30 ℃ of shaking table 250rpm cultivate 48h.Get 10mL first order seed substratum, be inoculated into the 500mL Erlenmeyer flask of 250mL secondary seed medium, 30 ℃ of shaking table 250rpm cultivate 24h.
(2) fermentation culture
Fermention medium: glucose 0.83%, CaCO
30.2%, soyflour 2.0%, yeast extract 0.1%, Fe (NH
4)
2SO
46H
2O 0.066%, and KCl 0.02%, MgSO
47H
2O 0.02%, FeSO
47H
2O0.0004%, Sag 471 (silicone antifoam agent) 0.02%, deionized water is supplied, and the pH value is 7.0.
Fermentation culture conditions: liquid amount is 30%, and the inoculation volume is 3%, air air flow 2L/min, and preceding 15h stirring intensity 240rpm, 370rpm then, 28 ℃ of culture temperature, fermentation back 10h keeps pH value 6.8 by adding ammoniacal liquor.Since the 28th hour, with every liter of nutrient solution per hour the amount of 0.26mL add aseptic caproic acid (50%v/v), until results.
(3) capric acid stream adds
The thalli growth initial stage, medicine irritation was relatively more responsive to external world, add capric acid this moment and be unfavorable for that thalli growth and daptomycin synthesize, monitor by high performance liquid chromatography, add capric acid in the logarithmic growth early stage (about 10.5h) that the daptomycin parent nucleus has just produced, certain herbaceous plants with big flowers acid final concentration is 0.4mmol/L, allow thalline adapt to growing environment, relatively help the synthetic of later stage daptomycin.Yet, once add precursor and easily cause the precursor deficiency, can not continue a large amount of synthetic daptomycins, therefore (36h) adds certain herbaceous plants with big flowers acid when thalline enters logarithmic phase, and certain herbaceous plants with big flowers acid final concentration is 0.9mmol, guarantees the precursor consumption of mass production daptomycin.
The fermentation unit that records daptomycin in the fermented liquid after fermentation reaction finishes is 2013mg/L.
Embodiment 3
(1) seed culture
The first order seed substratum: trypticase soya broth 4.0%, dextrin 3.0%, deionized water is supplied, and the pH value is 8.0.
Secondary seed medium: soyflour 0.5%, yeast extract 0.6%, calglucon 1.0%, KCl0.03%, MgSO
47H
2O 0.03%, FeSO
47H
2O 0.0005%, Sag 471 (silicone antifoam agent) 0.04%, and deionized water is supplied, and the pH value is 8.0.
Culture condition: bacterial classification recovery back direct inoculation is in the 250mL Erlenmeyer flask that 50mL first order seed substratum is housed, and 30 ℃ of shaking table 250rpm cultivate 48h.Get 10mL first order seed substratum, be inoculated into the 500mL Erlenmeyer flask of 250mL secondary seed medium, 30 ℃ of shaking table 250rpm cultivate 24h.
(2) fermentation culture
Fermention medium: glucose 0.80%, CaCO
30.1%, soyflour 1.0%, yeast extract 0.05%, Fe (NH
4)
2SO
46H
2O 0.065%, and KCl 0.015%, MgSO
47H
2O 0.015%, FeSO
47H
2O0.0003%, Sag 471 (silicone antifoam agent) 0.01%, deionized water is supplied, and the pH value is 8.0.
Fermentation culture conditions: liquid amount is 40%, and the inoculation volume is 4%, air air flow 3L/min, and preceding 15h stirring intensity 250rpm, 380rpm then, 30 ℃ of culture temperature, fermentation back 10h keeps pH value 7.0 by adding ammoniacal liquor.Since the 28th hour, with every liter of nutrient solution per hour the amount of 0.27mL add aseptic caproic acid (50%v/v), until results.
(3) capric acid stream adds
The thalli growth initial stage, medicine irritation was relatively more responsive to external world, add capric acid this moment and be unfavorable for that thalli growth and daptomycin synthesize, monitor by high performance liquid chromatography, add capric acid in the logarithmic growth early stage (about 11h) that the daptomycin parent nucleus has just produced, certain herbaceous plants with big flowers acid final concentration is 0.5mmol/L, allow thalline adapt to growing environment, relatively help the synthetic of later stage daptomycin.Yet, once add precursor and easily cause the precursor deficiency, can not continue a large amount of synthetic daptomycins, therefore (37h) adds certain herbaceous plants with big flowers acid when thalline enters logarithmic phase, and certain herbaceous plants with big flowers acid final concentration is 1.0mmol, guarantees the precursor consumption of mass production daptomycin.
The fermentation unit that records daptomycin in the fermented liquid after fermentation reaction finishes is 2041mg/L.
Embodiment 4
(1) seed culture
The first order seed substratum: trypticase soya broth 3.5%, dextrin 3.0%, deionized water is supplied, and the pH value is 7.5.
Secondary seed medium: soyflour 0.5%, yeast extract 0.6%, calglucon 1.2%, KCl0.03%, MgSO
47H
2O 0.03%, FeSO
47H
2O 0.0005%, Sag 471 (silicone antifoam agent) 0.04%, and deionized water is supplied, and the pH value is 7.5.
Culture condition: bacterial classification recovery back direct inoculation is in the 250mL Erlenmeyer flask that 50mL first order seed substratum is housed, and 30 ℃ of shaking table 250rpm cultivate 48h.Get 10mL first order seed substratum, be inoculated into the 500mL Erlenmeyer flask of 250mL secondary seed medium, 30 ℃ of shaking table 250rpm cultivate 24h.
(2) fermentation culture
Fermention medium: glucose 0.85%, CaCO
30.3%, soyflour 3.0%, yeast extract 0.15%, Fe (NH
4)
2SO
46H
2O 0.068%, and KCl 0.02%, MgSO
47H
2O 0.025%, FeSO
47H
2O0.0005%, Sag 471 (silicone antifoam agent) 0.03%, deionized water is supplied, and the pH value is 7.5.
Fermentation culture conditions: liquid amount is 30%, and the inoculation volume is 3%, air air flow 5L/min, and preceding 15h stirring intensity 260rpm, 400rpm then, 32 ℃ of culture temperature, fermentation back 10h keeps pH value 7.5 by adding ammoniacal liquor.Since the 28th hour, with every liter of nutrient solution per hour the amount of 0.28mL add aseptic caproic acid (50%v/v), until results.
(3) capric acid stream adds
The thalli growth initial stage, medicine irritation was relatively more responsive to external world, add capric acid this moment and be unfavorable for that thalli growth and daptomycin synthesize, monitor by high performance liquid chromatography, add capric acid in the logarithmic growth early stage (about 10.5h) that the daptomycin parent nucleus has just produced, certain herbaceous plants with big flowers acid final concentration is 0.6mmol/L, allow thalline adapt to growing environment, relatively help the synthetic of later stage daptomycin.Yet, once add precursor and easily cause the precursor deficiency, can not continue a large amount of synthetic daptomycins, therefore (38h) adds certain herbaceous plants with big flowers acid when thalline enters logarithmic phase, and certain herbaceous plants with big flowers acid final concentration is 1.1mmol, guarantees the precursor consumption of mass production daptomycin.
The fermentation unit that records daptomycin in the fermented liquid after fermentation reaction finishes is 2113mg/L.
Embodiment 5
(1) seed culture
The first order seed substratum: trypticase soya broth 3.8%, dextrin 2.8%, deionized water is supplied, and the pH value is 6.8.
Secondary seed medium: soyflour 0.48%, yeast extract 0.58%, calglucon 1.1%, KCl 0.03%, MgSO
47H
2O 0.03%, FeSO
47H
2O 0.0004%, Sag 471 (silicone antifoam agent) 0.03%, and deionized water is supplied, and the pH value is 6.8.
Culture condition: bacterial classification recovery back direct inoculation is in the 250mL Erlenmeyer flask that 50mL first order seed substratum is housed, and 30 ℃ of shaking table 250rpm cultivate 48h.Get 10mL first order seed substratum, be inoculated into the 500mL Erlenmeyer flask of 250mL secondary seed medium, 30 ℃ of shaking table 250rpm cultivate 24h.
(2) fermentation culture
Fermention medium: glucose 0.83%, CaCO
30.2%, soyflour 2.0%, yeast extract 0.12%, Fe (NH
4)
2SO
46H
2O 0.066%, and KCl 0.025%, MgSO
47H
2O 0.025%, FeSO
47H
2O0.0004%, Sag 471 (silicone antifoam agent) 0.03%, deionized water is supplied, and the pH value is 6.8.
Fermentation culture conditions: liquid amount is 50%, and the inoculation volume is 5%, air air flow 5L/min, and preceding 15h stirring intensity 280rpm, 430rpm then, 35 ℃ of culture temperature, fermentation back 10h keeps pH value 6.8 by adding ammoniacal liquor.Since the 28th hour, with every liter of nutrient solution per hour the amount of 0.28mL add aseptic caproic acid (50%v/v), until results.
(3) capric acid stream adds
The thalli growth initial stage, medicine irritation was relatively more responsive to external world, add capric acid this moment and be unfavorable for that thalli growth and daptomycin synthesize, monitor by high performance liquid chromatography, add capric acid in the logarithmic growth early stage (about 11.5h) that the daptomycin parent nucleus has just produced, certain herbaceous plants with big flowers acid final concentration is 0.7mmol/L, allow thalline adapt to growing environment, relatively help the synthetic of later stage daptomycin.Yet, once add precursor and easily cause the precursor deficiency, can not continue a large amount of synthetic daptomycins, therefore (40h) adds certain herbaceous plants with big flowers acid when thalline enters logarithmic phase, and certain herbaceous plants with big flowers acid final concentration is 1.2mmol, guarantees the precursor consumption of mass production daptomycin.
The fermentation unit that records daptomycin in the fermented liquid after fermentation reaction finishes is 2082mg/L.
Claims (4)
1. bacterial strain WKL-126 who produces daptomycin, its called after Streptomyces roseosporus (Streptomyces roseosporus) of classifying has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its deposit number is 3731.
2. method that adopts the described superior strain fermentation method of claim 1 to produce daptomycin is characterized in that:
The first order seed substratum is: trypticase soya broth 3.0%～5.0%, and dextrin 2.0%～4.0%, deionized water is supplied, and the pH value is 6.0～8.0.
Secondary seed medium is: soyflour 0.4%～0.6%, and yeast extract 0.4%～0.7%, calglucon 0.7%～1.3%, KCl 0.01%～0.03%, MgSO
47H
2O 0.01%～0.03%, FeSO
47H
2O0.0003%～0.0005%, Sag 471 (silicone antifoam agent) 0.02%～0.04%, deionized water is supplied, and the pH value is 6.0～8.0.
Fermention medium is: glucose 0.80%～0.85%, CaCO
30.1%～0.3%, soyflour 1.0%～3.0%, yeast extract 0.05%～0.15%, Fe (NH
4)
2SO
46H
2O 0.065%～0.068%, KCl0.015%～0.025%, MgSO
47H
2O 0.015%～0.025%, FeSO
47H
2O 0.0003%～0.0005%, Sag 471 (silicone antifoam agent) 0.01%～0.03%, and deionized water is supplied, and the pH value is 6.0～8.0.
3. method that adopts the described superior strain fermentation method of claim 1 to produce daptomycin, it is characterized in that, fermentation culture conditions is: liquid amount is 20%～50%, the inoculation volume is 2%～5%, air air flow 1～5L/min, preceding 15h stirring intensity 220～280rpm, 350～450rpm then, 25～35 ℃ of culture temperature, fermentation back 10h by add ammoniacal liquor keep pH value 6.5 or more than.Since the 28th hour, add aseptic caproic acid (50%v/v) with the amount of every liter of nutrient solution 0.25～0.28mL/L, until results.
4. method that adopts the described superior strain fermentation method of claim 1 to produce daptomycin, it is characterized in that, capric acid stream adds CONTROL PROCESS: the interpolation time of capric acid and interpolation concentration (the capric acid concentration of measuring in the fermented liquid) are about 10～12h, 0.1～0.8mmol/L and 35～40h, 0.8～1.2mmol/L, adopt the method for fed-batch to carry out the regulation and control of tunning.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703551A (en) * | 2012-06-27 | 2012-10-03 | 中国药科大学 | Novel method for adding decanoic acid in fermentation process of daptomycin |
| CN102796680A (en) * | 2012-07-04 | 2012-11-28 | 鲁南新时代生物技术有限公司 | Streptomyces roseosporus and method for producing daptomycin by utilizing combined precursor |
| CN111103373A (en) * | 2020-01-03 | 2020-05-05 | 丽珠集团福州福兴医药有限公司 | Daptomycin detection method |
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| WO2001005330A1 (en) * | 1999-07-20 | 2001-01-25 | Karram Mickey M | Apparatus and method to facilitate intermittent self-catheterization |
| CN1793356A (en) * | 2005-11-03 | 2006-06-28 | 天津大学 | Process for selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005330A1 (en) * | 1999-07-20 | 2001-01-25 | Karram Mickey M | Apparatus and method to facilitate intermittent self-catheterization |
| CN1793356A (en) * | 2005-11-03 | 2006-06-28 | 天津大学 | Process for selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete |
Non-Patent Citations (1)
| Title |
|---|
| 《安徽农业科学》 20081231 余继叁等 玫瑰孢链霉菌NRRL11379产达托霉素前体物A21978C的发酵培养基优化 7974-7976 第36卷, 第19期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703551A (en) * | 2012-06-27 | 2012-10-03 | 中国药科大学 | Novel method for adding decanoic acid in fermentation process of daptomycin |
| CN102796680A (en) * | 2012-07-04 | 2012-11-28 | 鲁南新时代生物技术有限公司 | Streptomyces roseosporus and method for producing daptomycin by utilizing combined precursor |
| CN102796680B (en) * | 2012-07-04 | 2018-06-05 | 鲁南新时代生物技术有限公司 | A kind of Streptomyces roseosporus and its method for producing Daptomycin using precursor is combined |
| CN111103373A (en) * | 2020-01-03 | 2020-05-05 | 丽珠集团福州福兴医药有限公司 | Daptomycin detection method |
| CN111103373B (en) * | 2020-01-03 | 2022-04-19 | 丽珠集团福州福兴医药有限公司 | Daptomycin detection method |
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